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Blood flow is adjusted to tissue demand through rapidly ascending vasodilatations resulting from conduction of hyperpolarisation through vascular gap junctions. We investigated how these dilatations can spread without attenuation if mediated by an electrical signal. Cremaster muscle arterioles were studied in vivo by simultaneously measuring membrane potential and vessel diameter. Focal application of acetylcholine elicited hyperpolarisations which decayed passively with distance from the local site,while dilatation spread upstream without attenuation. Analysis of simultaneous recordings at the local site revealed that hyperpolarisation and dilatation were only linearly related over a restricted voltage range to a threshold potential, beyond which dilatation was maximal. Experimental data could be simulated in a computational model with electrotonic decay of hyperpolarisation but imposition of this threshold. The model was tested by reducing the amplitude of the local hyperpolarisation which led to entry into the linear range closer to the local site and decay of dilatation. Serial section electron microscopy and light dye treatment confirmed that the spread of dilatation occurred through the endothelium and that the two cell layers were tightly coupled. Generality of the mechanism was demonstrated by applying the model to the attenuated propagation of dilatation found in larger arteries.We conclude that long distance spread of locally initiated dilatations is not due to a regenerative electrical phenomenon, but rather a restricted linear relationship between voltage and vessel tone, which minimises the impact of electrotonic decay of voltage. Disease-related alterations in endothelial coupling or ion channel expression could therefore decrease the ability to adjust blood flow to meet metabolic demand.

While L-type voltage-dependent calcium channels have long been considered the predominant source of calcium for myogenic constriction, recent studies of both cerebral and systemic circulations have provided evidence for the prominent expression of other members of the voltage-dependent calcium channel family, in particular the low voltage activated T-type channels. Although physiological studies have not supported the involvement of a classical low voltage activated, T-type channel in vascular function, evidence is accumulating that points to the involvement of a non-L-type, high voltage activated channel with sensitivity to T-type channel antagonists. We propose that this may arise due to expression of a T-type channel splice variant with unique biophysical characteristics resulting in a more depolarised profile. Expression of these channels in smooth muscle cells would broaden the voltage range over which sustained calcium influx occurs, while expression of T-type channels in endothelial cells could provide a feedback mechanism to prevent excessive vasoconstriction. Perturbation of this balance during pathophysiological conditions by upregulation of channel expression and endothelial dysfunction could contribute to vasospastic conditions and therapy-refractory hypertension.

1. Coordinated oscillations in diameter occur spontaneously in cerebral vessels and depend on the opening of voltage dependent calcium channels. However, the mechanism that induces the initial depolarisation has remained elusive. We investigated the involvement of canonical transient receptor potential (TRPC) channels, which encode nonselective cation channels passing Na(+) and Ca(2+) currents, by measuring changes in diameter, intracellular Ca(2+) and membrane potential in branches of juvenile rat basilar arteries. 2. Removal of extracellular Ca(2+) abolished vasomotion and relaxed arteries, but paradoxically produced depolarisation. 3. Decrease in temperature to 24 degrees C or inhibition of phospholipase C (PLC) abolished vasomotion, hyperpolarised and relaxed arteries and decreased intracellular Ca(2+). 4. Reduction in the driving force for Na(+) through decrease in extracellular Na(+) produced similar effects and prevented the depolarisation elicited by removal of extracellular Ca(2+). 5. Nonselective TRP channel blockers, SKF96365 and gadolinium, mimicked the effects of inhibition of the PLC pathway. 6. Depolarisation of vessels in which TRP channels were blocked with SKF96365 reinstated vascular tone and vasomotion. 7. Quantitative polymerase chain reaction revealed TRPC1 as the predominantly expressed TRPC subtype. 8. Incubation with a function blocking TRPC1 antibody delayed the onset of vasomotion. 9. We conclude that nonselective cation channels contribute to vasoconstriction and vasomotion of cerebral vessels by providing an Na(+)-induced depolarisation that activates voltage dependent calcium channels. Our antibody data suggest the involvement of TRPC1 channels that might provide a target for treatment of therapy-refractory vasospasm.

Intercellular communication through gap junctions coordinates vascular tone by the conduction of vasomotor responses along the vessel wall. Gap junctions in arterioles are composed of different connexins (Cxs) (Cx40, Cx37, Cx45, Cx43), but it is unknown whether Cxs are interchangeable. We used mice with a targeted replacement of Cx40 by Cx45 (Cx40KI45) to explore whether Cx45 can functionally replace Cx40 in arterioles. Arterioles were locally stimulated using acetylcholine, bradykinin, adenosine, and K + in the cremaster of Cx40KI45, Cx40-deficient (Cx40ko), and wild-type mice, and diameter changes were assessed by intravital microscopy. Additionally, arterial pressure was measured by telemetry and Cx expression verified by immunofluorescence. Acetylcholine initiated a local dilation of a similar amplitude in all genotypes (≈50%), which was rapidly conducted to upstream sites (1200 μm distance) without attenuation in wild type. In marked contrast, the remote dilation was significantly reduced in Cx40ko (25±3%) and Cx40KI45 (24±2%). Likewise, dilations initiated by bradykinin application were conducted without attenuation up to 1200 μm in wild type but not in Cx40ko and Cx40KI45. Adenosine-induced dilations and K + -induced constrictions were conducted similarly with decaying amplitude in all genotypes. Arterial pressure was strongly elevated in Cx40ko (161±1 versus 116±2 mm Hg) but only moderately in Cx40KI45 (133±8 mm Hg). This demonstrates that Cx40 function is critical for the conduction of acetylcholine and bradykinin dilations and cannot be substituted by Cx45. Therefore, unique properties of Cx40 are required for endothelial signal conduction, whereas nonspecific restoration of communication maintains additional functions related to blood pressure control.

A smooth muscle hyperpolarization is essential for endothelium-dependent hyperpolarizing factor–mediated dilations. It is debated whether the hyperpolarization is induced by a factor (endothelium-derived hyperpolarizing factor) and/or is attributable to direct current transfer from the endothelium via myoendothelial gap junctions. Here, we measured membrane potential in endothelial cells (EC) and smooth muscle cells (SMC) in vivo at rest and during acetylcholine (ACh) application in the cremaster microcirculation of mice using sharp microelectrodes before and after application of specific blockers of Ca 2+ -dependent K + channels (K Ca ). Moreover, diameter changes in response to ACh were studied. Membrane potential at rest was lower in EC than SMC (−46.6±1.0 versus −36.5±1.0mV, P P P Ca (charybdotoxin or iberiotoxin) abrogated ACh-induced hyperpolarizations in SMC but did not alter endothelial hyperpolarizations. In contrast, apamin, a blocker of small conductance K Ca abolished ACh-induced hyperpolarizations in EC and had only small effects on SMC. ACh-induced dilations were strongly attenuated by iberiotoxin but only slightly by apamin. We conclude that myoendothelial coupling in arterioles in vivo in the murine cremaster is weak, as EC and SMC behaved electrically different. Small conductance K Ca mediate endothelial hyperpolarization in response to ACh, whereas large conductance K Ca are important in SMC. Because tight myoendothelial coupling was found in vitro in previous studies, we suggest that it is differentially regulated between vascular beds and/or by mechanisms acting in vivo.

Atherosclerosis and hyperlipidemia impair endothelium-dependent nitric oxide (NO)-mediated dilations in conducting arteries. In addition to NO, the endothelium releases an endothelium-derived hyperpolarizing factor (EDHF) in response to acetylcholine (ACh), which is particularly important in microvessels and initiates a dilation that conducts along the vessel through gap junctional communication. The expression of connexins is, however, altered by hypercholesterolemia. Therefore, we studied endothelium-dependent dilations and their conduction in murine hypercholesterolemic models. Dilations were assessed by intravital microscopy in arterioles with a diameter of ∼35 µm in ApoE and LDL receptor (LDLR–/–)-deficient mice after superfusion or locally confined application of ACh. ACh induced comparable concentration-dependent dilations in wild-type, LDLR–/–, and ApoE–/– mice fed a normal or high-cholesterol diet, however EC50 was slightly higher in ApoE–/– mice. Furthermore, the NO donor sodium-nitroprusside dilated arterioles to a similar extent (∼60%). Locally initiated ACh dilations (∼68%) conducted up to a distance of 1,100 µm without significant attenuation even under severe hypercholesterolemic conditions. Since ACh dilation in the arterioles of mice is mainly mediated via EDHF, we conclude that hypercholesterolemia does not alter EDHF release and efficacy. This conclusion is confirmed by an intact conducted response since EDHF is a prerequisite for this response. The intact conduction also suggests that gap-junctional communication is functionally preserved in these models.

While L-type voltage-dependent calcium channels have long been considered the predominant source of calcium for myogenic constriction, recent studies of both cerebral and systemic circulations have provided evidence for the prominent expression of other members of the voltage-dependent calcium channel family, in particular the low voltage activated T-type channels. Although physiological studies have not supported the involvement of a classical low voltage activated, T-type channel in vascular function, evidence is accumulating that points to the involvement of a non-L-type, high voltage activated channel with sensitivity to T-type channel antagonists. We propose that this may arise due to expression of a T-type channel splice variant with unique biophysical characteristics resulting in a more depolarised profile. Expression of these channels in smooth muscle cells would broaden the voltage range over which sustained calcium influx occurs, while expression of T-type channels in endothelial cells could provide a feedback mechanism to prevent excessive vasoconstriction. Perturbation of this balance during pathophysiological conditions by upregulation of channel expression and endothelial dysfunction could contribute to vasospastic conditions and therapy-refractory hypertension.

Background— It has been proposed that activation of endothelial SK3 (K Ca 2.3) and IK1 (K Ca 3.1) K + channels plays a role in the arteriolar dilation attributed to an endothelium-derived hyperpolarizing factor (EDHF). However, our understanding of the precise function of SK3 and IK1 in the EDHF dilator response and in blood pressure control remains incomplete. To clarify the roles of SK3 and IK1 channels in the EDHF dilator response and their contribution to blood pressure control in vivo, we generated mice deficient for both channels. Methods and Results— Expression and function of endothelial SK3 and IK1 in IK1 −/− /SK3 T/T mice was characterized by patch-clamp, membrane potential measurements, pressure myography, and intravital microscopy. Blood pressure was measured in conscious mice by telemetry. Combined IK1/SK3 deficiency in IK1 −/− /SK3 T/T (+doxycycline) mice abolished endothelial K Ca currents and impaired acetylcholine-induced smooth muscle hyperpolarization and EDHF-mediated dilation in conduit arteries and in resistance arterioles in vivo. IK1 deficiency had a severe impact on acetylcholine-induced EDHF-mediated vasodilation, whereas SK3 deficiency impaired NO-mediated dilation to acetylcholine and to shear stress stimulation. As a consequence, SK3/IK1-deficient mice exhibited an elevated arterial blood pressure, which was most prominent during physical activity. Overexpression of SK3 in IK1 −/− /SK3 T/T mice partially restored EDHF- and nitric oxide-mediated vasodilation and lowered elevated blood pressure. The IK1-opener SKA-31 enhanced EDHF-mediated vasodilation and lowered blood pressure in SK3-deficient IK1 +/+ /SK3 T/T (+doxycycline) mice to normotensive levels. Conclusions— Our study demonstrates that endothelial SK3 and IK1 channels have distinct stimulus-dependent functions, are major players in the EDHF pathway, and significantly contribute to arterial blood pressure regulation. Endothelial K Ca channels may represent novel therapeutic targets for the treatment of hypertension.

Aims The activation of endothelial Ca2+-dependent K+-channels, KCa3.1 (IKCa), and KCa2.3 (SKCa) has been proposed to be a prerequisite for endothelial hyperpolarization, which subsequently hyperpolarizes and relaxes smooth muscle [endothelium-derived hyperpolarizing factor (EDHF)-type dilation] and initiates conducted dilations. Although EDHF is the main mediator of acetylcholine (ACh)-induced dilation in the murine skeletal microcirculation, the differential contribution of KCa3.1 and KCa2.3 is not known. Methods and results We assessed agonist-induced and conducted dilations as well as endothelial hyperpolarization in the cremaster microcirculation of KCa3.1-deficient (KCa3.1−/−) and wild-type mice (wt) in vivo after blockade of NO and prostaglandins. Compared with wt, resting tone was enhanced by ∼25% in arterioles of KCa3.1−/− mice. ACh-induced dilations in KCa3.1−/− mice were virtually abolished at low and intermediate concentrations and a remaining dilation at 10 µmol/L ACh was abrogated by blockade of KCa2.3 with UCL1684. Sodium nitroprusside- and adenosine-induced dilations were similar in wt and KCa3.1−/−. Focal application of ACh induced dilations at the local site in both genotypes, which conducted along the vessel. However, the amplitude of the dilation decreased with distance only in KCa3.1−/−. Blockade of KCa2.3 in wt did not affect conducted dilations. A KCa3.1 opener induced a conducting dilation in wt but not in KCa3.1−/−. Membrane potential recordings in vivo demonstrated endothelial hyperpolarization in response to ACh in both genotypes; however, the hyperpolarization was severely impaired in KCa3.1−/− (Δ membrane potential: −3 ± 1 vs. −14 ± 2 mV). Conclusion We conclude that KCa3.1 is of major importance for endothelial hyperpolarization and EDHF-type responses in skeletal muscle arterioles, and its deficiency is not compensated by KCa2.3. Sole activation of KCa3.1 is capable of initiating conducted responses, and KCa3.1 may contribute to the propagation of the signal, although its presence is not mandatory.

Gap junctions are formed in the cardiovascular system by connexin40 (Cx40), Cx37, Cx43, and Cx45. These low resistance channels allow the transfer of ions and small molecules between cells. The longitudinal coupling of endothelial and smooth muscle cells via gap junctions allows the spread of changes in membrane potential along the vascular wall and hence provides conduction pathways within the vessel itself. Functionally, this tight coupling is reflected by the spread of locally initiated vasomotor responses along the arteriole which are termed conducted responses. Conducted dilations are initiated by the application of endothelium-dependent stimuli which result in local hyperpolarization. This signal spreads along the wall, most likely along the endothelial cell layer, to elicit a coordinated dilation of the arteriole over a considerable distance. Likewise, the opposite signal (depolarization) spreads along the vessel giving rise to a conducted constriction. The latter response is however most likely transmitted along the smooth muscle cell layer. Thus, conducted responses reflect the synchronized behavior of the cells of the vascular wall. It is assumed that conducted responses are critical for the matching of oxygen delivery and tissue needs because they contribute to an ascending dilation which lowers resistance along the length of the arterioles and upstream vessels in a well-tuned fashion. Herein, Cx40 is of special importance because it is critically required for intact signal transduction along the endothelial cell layer. In addition, Cx40 mediates pressure feedback inhibition on renin synthesis in the kidney. Both, vascular and renal function of Cx40, may be involved in the hypertension that is observed in Cx40-deficient animals. In this review, we will summarize physiologic function of connexins in arterioles and briefly address their role in the kidney with respect to renin secretion.

The endothelium plays a key role in the control of vascular tone and alteration in endothelial cell function contributes to several cardiovascular disease states. Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF signaling is thought to be initiated by activation of endothelial Ca 2+ -activated K + channels (K Ca ), leading to hyperpolarization of the endothelium and subsequently to hyperpolarization and relaxation of vascular smooth muscle. In the present study, we tested the functional role of the endothelial intermediate-conductance K Ca (IK Ca /K Ca 3.1) in endothelial hyperpolarization, in EDHF-mediated dilation, and in the control of arterial pressure by targeted deletion of K Ca 3.1. K Ca 3.1-deficient mice (K Ca 3.1 −/− ) were generated by conventional gene-targeting strategies. Endothelial K Ca currents and EDHF-mediated dilations were characterized by patch-clamp analysis, myography and intravital microscopy. Disruption of the K Ca 3.1 gene abolished endothelial K Ca 3.1 currents and significantly diminished overall current through K Ca channels. As a consequence, endothelial and smooth muscle hyperpolarization in response to acetylcholine was reduced in K Ca 3.1 −/− mice. Acetylcholine-induced dilations were impaired in the carotid artery and in resistance vessels because of a substantial reduction of EDHF-mediated dilation in K Ca 3.1 −/− mice. Moreover, the loss of K Ca 3.1 led to a significant increase in arterial blood pressure and to mild left ventricular hypertrophy. These results indicate that the endothelial K Ca 3.1 is a fundamental determinant of endothelial hyperpolarization and EDHF signaling and, thereby, a crucial determinant in the control of vascular tone and overall circulatory regulation.

Communication between cells is important to the microcirculation and enables the coordination of cellular behavior along the length of the vessel. Arterioles span considerable distances within the microcirculatory network, and thus flow changes require the adaptation of vessel resistance over the whole length of the vessel in order to be effective. Such a task requires communication along the vessel wall, and gap junction channels that connect endothelial as well as smooth muscle cells with each other set the stage for this requirement. Communication along the vessel wall can be tested experimentally by confined local stimulation of arterioles either in vitro or in vivo. Certain vascular stimuli induce both a local response and a concomitant uniform remote response, confirming the rapid conduction of vasomotor stimuli along the vessel wall. Gap junctions in vascular tissue are composed of connexins (Cx) Cx40, Cx43, Cx37 and Cx45. Of these, Cx40 is of special importance: its lack results in a deficient conduction of vasodilator stimuli along the vessel wall. Interestingly, Cx40-deficient mice display an elevated mean arterial pressure, suggesting that Cx40-depending gap junctional coupling is necessary to regulate vascular behavior and peripheral resistance. While the role of other connexins is less well established, an abundance of experimental data has proven the necessity of gap junctional communication to coordinate vascular behavior during adaptive blood flow regulation.

Communication between cells is important to the microcirculation and enables the coordination of cellular behavior along the length of the vessel. Arterioles span considerable distances within the microcirculatory network, and thus flow changes require the adaptation of vessel resistance over the whole length of the vessel in order to be effective. Such a task requires communication along the vessel wall, and gap junction channels that connect endothelial as well as smooth muscle cells with each other set the stage for this requirement. Communication along the vessel wall can be tested experimentally by confined local stimulation of arterioles either in vitro or in vivo. Certain vascular stimuli induce both a local response and a concomitant uniform remote response, confirming the rapid conduction of vasomotor stimuli along the vessel wall. Gap junctions in vascular tissue are composed of connexins (Cx) Cx40, Cx43, Cx37 and Cx45. Of these, Cx40 is of special importance: its lack results in a deficient conduction of vasodilator stimuli along the vessel wall. Interestingly, Cx40-deficient mice display an elevated mean arterial pressure, suggesting that Cx40-depending gap junctional coupling is necessary to regulate vascular behavior and peripheral resistance. While the role of other connexins is less well established, an abundance of experimental data has proven the necessity of gap junctional communication to coordinate vascular behavior during adaptive blood flow regulation.

Cellular interaction in vessels is achieved by multiple communication pathways, including gap junctions (GJs). They provide intercellular channels, allowing direct interaction of endothelial and smooth muscle cells and the coordination of cellular behaviour along the vessel. The latter is a prerequisite for large flow increases because an adaptation of resistance along the vessel length is required. Longitudinal communication is studied by confined local stimulation of arterioles and the observation of responses at distant locations. Certain vascular stimuli induce local and concomitant remote responses of a similar type, verifying rapid longitudinal conduction of vasomotor signals, most likely changes in membrane potential. This is achieved for dilatory responses via the endothelium, possibly by an endothelium-derived hyperpolarising factor (EDHF) that induces local hyperpolarisation, which is then transferred to remote sites through GJs. In vessels, GJs are composed of different connexins (Cx), but Cx40 is of special importance because its lack impairs longitudinal conduction of vasodilations. Interestingly, Cx40-deficient mice are hypertensive, suggesting that Cx40-dependent coupling is necessary to regulate vascular behaviour and peripheral resistance. While the role of other connexins is less well established, an abundance of data has proven the necessity of GJ communication to coordinate vascular behaviour during blood flow regulation.